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In situ hybridization experiments on human chromosomes were performed using probes corresponding to the 5' and 3' parts of human TSHR cDNA. Both probes allowed a regional localization on chromosome 14q31.

Differential hybridization of cDNAs corresponding to mRNAs expressed in the human endometrium during the secretory phase or during the first trimester of pregnancy, but not during the proliferative phase, allowed us to isolate and characterize cDNAs encoding human placental protein 14 (PP14). The cDNA was used to isolate the PP14 gene from a human genomic library. The entire gene encompasses 5.05 kb divided into seven exons by six introns. The human PP14 gene shows identical organization with the ovine beta-lactoglobulin gene, as expected from protein homology. Sequencing of 3 kb of the 5'-flanking region of the gene allowed us to characterize a 400-bp duplication of the PP14 gene lying at position -2,660. This duplication was homologous to 100 bp of exon 4 and 300 bp of intron 4, including 180 bp corresponding exactly to the right arm of an Alu element lying on the complementary strand. This homology suggests that this duplication may have arisen through a retroposition event.

Several vectors were used to express the complementary DNA for breast cancer estrogen-induced protein BCEI (also called pS2) in Escherichia coli. The best results were obtained by using the pUR 290 expression vector after deletion of the sequence encoding the signal peptide of the protein. In these conditions, beta-galactosidase-BCEI/pS2 fusion protein accounted for approximately 20% of total proteins in bacterial extracts. It was purified by chromatography on DEAE-Trisacryl or by gel electrophoresis and electroelution. Polyclonal antibodies were obtained by immunization of rabbits and goats, and monoclonal antibodies were raised in mice. Two types of monoclonal antibodies were obtained: one class recognized the native protein and was very efficient for the immunoprecipitation and immunopurification of the protein from breast cancer cells; a second class recognized the denatured protein and was especially effective for immunoblot studies. BCEI/pS2 could be detected by immunocytochemistry in breast cancer biopsies using monoclonal antibodies on frozen or paraffin-embedded sections. One of the antibodies (mBCEI11) exhibited high affinity for the protein and could be used at 1.9 micrograms/ml concentration for immunolabeling of histological sections. The mBCEI11 antibody was used in immunoaffinity chromatography to purify the peptide in a single step from culture media of estrogen-treated MCF-7 cells.

Over 90 mouse monoclonal antibodies have been raised against rabbit and human uterine progesterone receptor (PR). These antibodies, because of their specificity, are powerful tools with which to examine the localization, structure, and function of PR. A selection of 22 well characterized mABs was made to test their ability to give the best immunocytochemical staining of PR in various species. Comparative analysis of the antibodies led to the following conclusions. Li 417 (and, to a lesser extent, Let 126) was the best monoclonal in humans; Let 126 and Mi 60 were the most sensitive monoclonals in guinea pigs, rabbits, and monkeys. In sheep, sows, cows and mares as well as in rats and chickens Let 81 or Let 548 gave the best results (Let 126 was also effective in sows and mares, while Li 169 was also effective in sheep and cows). Two antibodies (Li 169 and Let 548) cross-reacted with PR in all of the species tested, including mammals and birds, and appeared to recognize two highly conserved antigenic sites. Remarkably, these conserved sequences are located in the highly variable N-terminal part of the receptor; they may, thus, be related to the still poorly understood function of this domain of the receptor.

Complementary cDNA clones encoding the TSH (thyroid stimulatory hormone) receptor were isolated from a human thyroid lambda gt10 library using Iow stringency hybridization with LH/hCG (luteinizing hormone-human choriogonadotropic hormone) receptor probes. Sequencing of the clones showed a 764 amino acid open reading frame. The first 21 amino acids probably correspond to a signal peptide, the mature protein thus contains 743 amino acids (calculated molecular weight: 84,501 daltons). Its putative structure consists of a 394 amino acid extracellular domain, a 266 amino acid membrane spanning domain with 7 putative transmembrane segments and a 83 amino acid intracellular domain. A high degree of homology is observed with LH/hCG receptor suggesting the definition of a new subfamily of G-protein coupled receptors. Computer search showed the presence in the putative third intracellular loop of a motif resembling that described in the non receptor type protein tyrosine kinases (c-src, c-yes, c-fgr, etc...). RNA blots showed that the receptor messenger RNA consists of two major species of 4300 and 3900 nucleotides. The cDNA was inserted into an expression vector and after transfection into COS 7 cells it was shown to produce a functional TSH receptor.

Probes corresponding to human and porcine LH (luteinizing hormone) receptor cDNA were used for in situ hybridization to human chromosomes. This allowed us to assign the LH receptor gene to chromosome 2p21.

Radioautograms were made of uterine horns, cervix and vagina from guinea pigs injected with[3H]progesterone at various periods of the estrous cycle. The concentration of silver grains peaked at proestrus and estrus and then fell during metestrus, attaining a minimum at diestrus. These variations were not due to masking of binding sites by endogenous hormone since ovariectomy of guinea pigs at diestrus did not change this pattern. During proestrus and estrus, radioactivity was found in all parts of the endometrium and myometrium of the uterine horns. However, the muscle fibers and the cells of the stroma contained more silver grains than did the luminal and glandular epithelium. In the vagina, radioactivity was concentrated mostly in the nuclei of the basal layer cells of the stratified epithelium. During metestrus and diestrus, less radioactivity was present in these regions. These differences in labelling were observed simultaneously in all the uterine and vaginal cell types, suggesting that similar hormonal mechansims may control receptor variations in these different target cells.

7 alpha- and 7 beta-carboxymethyl-derivatives of 17-hydroxyprogesterone and 11-deoxycortisol have been synthesized. After coupling to bovine serum albumin, they were used to elicit antibodies in rabbits. No major difference in the steroid specificity of the antisera was observed when either 7 alpha- or 7 beta-epimers were used for immunization. In both cases, highly specific antisera were obtained which may possibly be used to assay human plasma 17-hydroxyprogesterone and 11-deoxycortisol without chromatographic purification.

A radioimmunoassay method to measure progesterone receptor in rabbit and human tissues was devised and applied to human breast cancer. A specific progesterone receptor antibody was prepared by purifying rabbit receptor by immunoaffinity chromatography, sodium dodecylsulphate-polyacrylamide gel electrophoresis and injection of the isolated 110,000 dalton receptor band into a goat. Immunoblot studies of progesterone target and non-target tissues showed the specificity of the antibody, which was used at a dilution of 1/45,000. The tracer consisted of 125I-labelled electroeluted 110,000 dalton receptor. The sensitivity of the method was 1 fmol/tube for the rabbit receptor and 3 fmol/tube for the human receptor. The intra-assay coefficient of variation was 11% for tumours positive for the progesterone receptor and 9.9% for those on the borderline (10-30 fmol receptor/mg protein). The interassay coefficients of variation were 20 and 19% respectively. The correlation between the radioimmunoassay and a steroid-binding assay was studied in 40 tumour biopsies. In 39 cases, very good correlation was found (r = 0.99); in a single case an immunoreactive protein was detected which apparently bound steroid poorly. One important feature of this method was that receptor immunoreactivity remained unchanged when either the tissue or the cytosol was exposed to a temperature of 20 degrees C for relatively long periods of time. Under the same conditions the steroid-binding capacity declined rapidly. This characteristic of the radioimmunoassay may prevent errors due to improper handling of tissue samples. Such stability was not observed for oestrogen receptors when measured by a sandwich immunoenzymatic method after incubation of tissue at 20 degrees C.

Mouse hybridomas secreting monoclonal antibodies against rabbit uterine progesterone receptor (PR) have been prepared. Several of these immunoglobulins exhibited high affinity towards human progesterone receptor and two (LET 126 and LET 64) were selected as giving the best immunoperoxidase staining of human progesterone target organs. Using the indirect peroxidase-antiperoxidase method of Sternberger, optimal conditions for demonstrating PR involved brief fixation of frozen sections with formaldehyde-containing fixatives, among them picric acid-paraformaldehyde. This method allowed us to detect the receptor in breast carcinoma epithelial cells, T47D cell line, and uterine endometrium and myometrium. No staining was observed in intestine and muscle. Specific staining for PR was confined to the nucleus, irrespective of the concentration of progesterone in the blood of the patient. In a preliminary study of 27 human breast cancers by the immunocytochemical method, the presence or absence of nuclear staining for PR correlated well with the concentration of cytosolic progesterone receptor determined by a steroid-binding assay on tumor extracts. Differences in the intensity and distribution of staining within a section were observed, suggesting heterogeneity of the PR content of breast cancer cells. In 19 tumors, the immunocytochemical method for PR localization was also used in combination with a slightly modified Abbott ER-ICA staining for estrogen receptor to compare the distribution of both receptors within the same biopsy on adjacent frozen sections. Various combinations of estrogen receptor and PR contents that have been determined by steroid-binding assay have also been detected by the double immunocytochemical assay.

The ultrastructure of 47 primary breast cancers was studied. For each tumour, the characteristics of approximately 200 malignant cells were examined. Eleven features were scored from 1 to 3 and multivariate analysis showed that seven of these could be used to define an ultrastructural index of differentiation. Differentiation at the ultrastructural level was associated with the presence of steroid receptors. Differentiated tumoral cells contained oestrogen receptor and progesterone receptor in 81.8% and 66.7% of cases, respectively. Poorly differentiated cells contained oestrogen receptors in 50% and progesterone receptor in 14.3% of cases. Comparison of histological grading by the method described by Scarff, Bloom & Richardson with the ultrastructural index of differentiation showed a rather loose correlation which was not significant in this group of 47 patients. The authors conclude that differentiation at tissue or cellular levels yields differing information, only the latter being closely correlated with steroid receptor presence.

Messenger RNAs (mRNAs) were prepared from MCF-7 breast cancer cells grown in the presence of estradiol. Complementary DNAs (cDNAs) were inserted into pBR322 plasmid and a library of 4400 recombinant bacterial clones was prepared. The clones were screened by in situ differential hybridization with cDNAs prepared from RNAs of MCF-7 cells grown either in the presence or absence of estradiol. Several estrogen-induced or estrogen-repressed clones were identified. One of them corresponded to a relatively frequent mRNA (0.8% of recombinant plasmids) of 650 nucleotides. The concentration of this mRNA was increased by estradiol (half maximal induction approximately 0.05 nM) but not by progesterone, dexamethasone, or dihydrotestosterone. Tamoxifen inhibited the effect of estradiol but was devoid of any agonistic activity when administered separately. This messenger was present in biopsies of breast cancer, but not in endometrium or liver. The cloned cDNA was sequenced. An open reading frame was found corresponding to a protein of less than 100 amino acids. A search of data banks showed no identity or marked similarity to previously published DNA or protein sequences, particularly to those of growth factors evoked by some characteristics of the coded polypeptide. The cloned cDNA probe was used to screen a library of Charon 4A phage containing human genomic fragments. Screening of 300,000 phages yielded two different recombinants hybridizing to the cDNA. Southern blot experiments using DNA from recombinant phage, MCF-7 cells, and placenta showed the presence of a unique gene exhibiting a similar restriction pattern in DNAs from malignant and nonmalignant tissues.

Uteroglobin has been studied under two aspects: 1) as a model of specific interaction between a protein and a steroid hormone: crystals were obtained and analyzed by X-ray diffraction; 2) as a marker of progesterone action in the endometrium: messenger RNA was translated, purified and transcribed into complementary DNA.

A mouse was immunized with purified rabbit uterine cytosolic progesterone receptor (specific activity: 3 nmol of steroid bound per mg of protein). After fusion of its spleen cells with Sp2-OAg myeloma cells, supernatants of 11 hybrid cultures were found to react in both an immunoenzymatic test and a double-immunoprecipitation test with the progesterone receptor. Clones were obtained from the five hybrid cells that gave the strongest response in both tests. Antibodies from cell culture supernatants and ascitic fluids were characterized. Three are of the IgG1 and two of the IgG2a isotype. Their apparent affinity for the progesterone receptor was measured by immunoprecipitation in physiological salt conditions. The equilibrium dissociation constants were between 0.1 and 4 nM. All five monoclonal antibodies crossreacted with the rabbit nuclear receptor, the human cytosolic receptor, and other mammalian (rat, guinea pig) but not avian (chicken) cytosolic progesterone receptors. There was no interaction with the glucocorticoid receptor and corticosteroid binding globulin.

Estrogen-binding proteins were observed in the cytosol of human pituitary adenomas. The Kd for estradiol was 0.44 nM at 0 C, and hormonal specificity was that expected for estrogen receptors. Sucrose gradient sedimentation experiments revealed that receptors from different tumors existed in either the 8S or the 4S form or both. Of the 23 tumors examined, 14 contained estrogen receptors. Receptors were more often present and their concentration was higher in PRL-secreting adenomas (mean +/- SEM, 20.6 +/- 13.4 fmol/mg proteins; 7 determinations) than in GH-secreting adenomas (1.4 +/- 0.8 fmol/mg protein; 9 determinations) and chromophobe tumors (4.1 +/- 1.6 fmol/mg protein; 7 determinations). There was also a correlation between the presence of estrogen receptors and histological signs of cell proliferation and tumor growth.

A method devised previously to precisely measure the concentration of unbound cortisol was used to compare plasma and cerebrospinal fluid (CSF) in 34 patients. In CSF the percentage of free cortisol was 88.4 +/- 6.3% (mean +/- SD). Its concentration was 4.94 +/- 2.00 ng/ml, only one third of the concentration of unbound cortisol in plasma of the same patients (14.3 +/- 8.8 ng/ml). Total and unbound cortisol in CSF were correlated with unbound cortisol in plasma; however, this correlation was rather loose (r = 0.527 and 0.554, respectively) due to large individual variations. Moreover, at high concentrations of unbound cortisol in plasma, the ratio of total cortisol in CSF/unbound cortisol in plasma was decreased. Thus, it is impossible to simply consider cortisol in CSF as a dialyzate of cortisol in plasma. The binding of cortisol in CSF was due to a protein having the electrophoretic mobility in polyacrylamide gels of plasma corticosteroid-binding globulin (CBG), and displaying the same hormonal specificity. The concentration of this protein was measured in 16 individual patients. This concentration, when expressed per protein content, was about two thirds of that of plasma CBG, and this ratio was extremely variable in individual patients. Individual variations of cortisol-binding globulin in CSF could not be attributed to variations of CBG in plasma nor to variations of protein content in CSF. There was an inverse relationship (r = 0.888) between unbound cortisol in CSF and the concentration of cortisol-binding globulin in this fluid, showing that CBG exerts a physiological role in CSF.

Many technical problems are encountered when measuring estradiol and progesterone receptors in breast tumors biopsies. They are due to both intrinsic characteristics of the tissue, and to receptors instability. Suggestions are made to establish an internal quality control in each laboratory at the different steps: obtention and storage of biopsies, purity checking of the reagents, control of the assay protocol, and calculations. Several methods to prepare control pools have been proposed. They use either mammary tumors or animal target organs, and stable concentrations of receptors have been obtained by storage in liquid nitrogen or lyophilization. Published studies have demonstrated important interlaboratory variations. These discrepancies might be in part reduced by using the same assay protocol in the different participating laboratories.

cDNA clones corresponding to the 3' and 5' non coding regions of the rabbit progesterone receptor (rPR) mRNA and genomic clones corresponding to the promoter and 5' flanking region of this gene were isolated and sequenced up to nucleotide -2761. The 3' non coding region is very long (3058-3553 nucleotides) and contains three different polyadenylation sites. Primer extension experiments and S1 mapping showed the existence of 2 transcription initiation sites 699 and 712 bp upstream from the initiator ATG. The promoter region contains two modified TATA boxes: TAGAAA at -17 and TAGA at -37bp. A CAACT sequence is present at position -100 and one consensus binding site for the transcription factor Sp1 is found at position -51. A 317 bp sequence was observed (positions -2590 to -2273) which belongs to the C family of the short interspersed repeats of the rabbit. Sequences resembling the consensus for estrogen and progesterone responsive elements are observed at several locations in the 5' flanking region. The progesterone receptor is present in tissue extracts mainly as a mixture of two molecular species (110 and 79 kDa) whose origin remains currently debated. By Northern blot analysis we have shown, using rabbit and human mRNAs, that these receptor species are not derived from separate mRNAs. Transcription-translation experiments also showed that, at least in vitro, they are not derived by use of different translation initiation sites on the same messenger RNA.

The presence of progesterone receptors was found to be associated with a favorable prognosis in 98 patients with primary breast cancer. The occurrence of metastases was 3.6 times less probable in patients with progesterone receptor-positive tumors than in patients with progesterone receptor-negative tumors. There was also an inverse relationship between the concentration of progesterone receptor and the frequency of metastases. However, there was no statistical correlation between frequency of local recurrences and progesterone receptor content of the tumor. In patients displaying clinical or histological criteria of gravity, the presence of progesterone receptors allowed us to define subgroups with good prognosis. Thus, in women with progesterone receptor-positive cancers, metastases had occurred at 18 months, in only 5% of the 39 Grade III cancers and in none of the 25 cases with invaded axillary nodes. Measurement of estradiol receptor (105 patients including the previous 98 patients) was found to be less effective for guiding the prognosis of early breast cancer. Combined evaluation of estradiol and progesterone receptors did not provide any more information than did the determination of progesterone receptor alone.

A simplified method is described for the assay of cortisol in human plasma. It is based upon tryptic hydrolysis of cortisol binding proteins in the plasma followed by radioimmunoassay. Only 1 microliter of plasma is used, no extraction of the hormone is necessary and most of the procedure is amenable to automatization. The sensitivity of the method is 10 pg, the coefficient of variation is 8.7% intrassay and 11.5% in different assays. The accuracy and the specificity were also verified. Comparison of this simplified technique with a standard method (extraction by methylene chloride and radiocompetition with corticosteroid binding globulin) gave a correlation coefficient (r) of 0.964.

The solubilized ("cytosolic") receptor present in the rabbit uterus in the absence of hormone and the chromatin-bound ("nuclear") receptor obtained after injection of a progestin were compared. Crude cellular extracts were analyzed by immunoblotting and receptors were purified by immunoaffinity chromatography. With both methods it was observed that the electrophoretic mobility of the "nuclear" receptor was slower than that of the "cytosolic" receptor. This difference in mobility appeared to be due to the existence of variably phosphorylated forms of receptor. The phosphorylation reaction was examined in uterine slices. In the absence of hormone the cytosolic receptor was phosphorylated. When hormone was added the phosphorylation of receptor was markedly enhanced and the electrophoretic mobility of the "nuclear" receptor was decreased. These experiments thus show that the receptor in its "cytosolic" form is a phosphoprotein. Under the effect of the hormone the receptor is further phosphorylated on some supplementary site(s). This polyphosphoprotein is the chromatin-bound, putatively active, form of the receptor. In this respect the intracellular progesterone receptor is similar to various membrane receptors for hormones and growth factors which are phosphorylated upon binding of their ligand.

[3H]Pregn-4-ene-3,20-dione ([3H]progesterone)-receptor complexes from human mammary carcinoma were found to be stabilized in the presence of glycerol. The dissociation rate constant was lowered and the equilibrium dissociation constant was decreased (KD=3 nM in the absence of glycerol and 1.1 nM in the presence of 30% glycerol), whereas no clear-cut effect on the association rate was observed and no change occurred in the concentration of binding sites. Cortisol was found to compete with [3H]progesterone only at concentrations higher than 1 muM. This made it possible to distinguish [3H]progesterone binding to the receptor from binding to corticosteroid-binding globulin. Synthetic progestins [6-chloro-17-acetoxypregna-4,6-diene-3,20-dione (chlromadinone acetate), 17alpha-ethinyl, 17-hydroxyestr-4-en-3-one (norethisterone), and 17,21-dimethyl-19-norpregna-4,9-diene-3,20-dione (R5020)] were found to have a high affinity for the receptor, whereas 5alpha-pregnane-3,20-dione had an affinity about one-half that of progesterone itself 5beta-Pregnane-3,20-dione, 17alpha-hydroxypregn-4-ene-3,20-dione (estradiol), 11beta,21-dihydroxy-pregn-4-ene-3,20-dione (corticosterone), estra-1,3,5(10)-triene-3,17beta-diol, and 17beta-hydroxyandrost-4-en-3-one (testosterone) were weak inhibitors of [3H]progesterone binding. Sedimentation on glycerol gradients showed different patterns in different tumors; i.e., [3H]progesterone specific binding having the characteristics of receptor was found either in the 8 S region, in the 4.5 S region, or in both. Activated progesterone-receptor complex from human mammary carcinoma cytosol was shown to bind to human DNA. An assay of the receptor based on these binding properties is described. This assay measures the total concentration of cytosol receptor since it makes possible the exchange of endogenous hormone for excess added [3H]progesterone. Of 55 biopsies examined by this method, 35 (64%) had a concentration of progesterone receptor-binding sites higher than 10 fmoles/mg protein. There was a positive correlation between the amounts of estrogen and progesterone receptors.

The complex of [3H]dexamethasone and rat liver receptor binds to rat liver DNA. This interaction takes place only in the presence of hormone and is enhanced by 'activation'. No evidence of saturatability can be obtained with concentrations of steroid-receptor complexes corresponding to those observed physiologically in the intact liver cell. The binding is inhibited by high ionic strength and by millimolar concentrations of divalent cations. No species specificity has been observed: the complex binds equally well to prokaryotic and eukaryotic DNA'S. There was no difference between binding to native and denatured DNA. In comparable conditions twice as much [3H]dexamethasone-receptor complexes were bound by DNA than by rat liver nuclei. Thus, the interaction of steroid-receptor complexes with DNA probably does not correspond to the recognition of a few very specific sequences. It is however possible that this interaction is actually operating in vivo in the intact cell.

A method for measuring the unbound fraction of plasma cortisol which is suitable for clinical use is described. It is shown that unless some precise experimental conditions are fulfilled, important errors may result when determining the unbound fraction of the hormone. The assay is practical (2 ml of plasma are necessary) and reproducible (coefficient of variation: 2.7% within the same assay and 3.1% in different assays). Unbound cortisol measured at 0800 h in 86 healthy individuals was 9.7 +/- 2.6% (SD) of total cortisol and 15.0 +/- 8.5 ng/ml of plasma (temperature: 37 C). No significant difference was found between men and women or according to age. In most cases, variations of unbound plasma cortisol were more important than the variations of total plasma cortisol. This explains why unbound cortisol was a better discriminator in some pathological conditions. In Cushing's syndrome unbound cortisol was found to be increased on the average 2.9 fold whereas total cortisol was only increased by 53%. Unbound cortisol was especially high (24.7 +/- 3.8% of total cortisol and 78 +/- 18.5 ng/ml of plasma) in Cushing's syndrome due to adrenal carcinoma. In adrenal insufficiency, unbound cortisol averaged 6% of total cortisol and 1.4 ng/ml of plasma.

A very high capacity immunoaffinity matrix for the purification of progesterone receptor was prepared by cross-linking a monoclonal antireceptor antibody to protein A-Sepharose through the Fc fragment. The monoclonal antibody was selected for its property of losing affinity for the receptor at pH 10.5, i.e., in conditions where the receptor remains stable for extensive periods of time. This made it possible to elute active receptor form the immunosorbent. From crude rabbit uterine cytosol the steroid-receptor complexes were purified in a single step. A 1-mL column (containing 7 mg of monoclonal antibody) bound 1600 pmol of steroid-receptor complexes of which 79.5% were eluted. The overall yield of purification was 49%. The specific activity of the purified steroid-receptor complexes was 6.71 +/- 0.79 nmol of bound steroid/mg of protein (mean +/- SE of four experiments). The purified receptor consisted of a mixture of 110 000- and 79 000-dalton forms. The latter appeared to be produced by proteolysis of the larger form during purification since immunoblot experiments showed that, at the start of purification, the 110 000-dalton form was present in overwhelming majority (80-95%) in the uterine cytosol and that the 79 000-dalton form only appeared during purification. This conclusion was also supported by the peptide analysis of both forms of receptor: the purified receptor was denatured and labeled with 125I; the 110 000- and 79 000-dalton forms were isolated by gel electrophoresis in denaturing conditions and electroelution and were then submitted to mild or extensive digestions by trypsin, chymotrypsin, and protease V8 from Staphylococcus aureus.(ABSTRACT TRUNCATED AT 250 WORDS)

Twenty-six patients with advanced breast cancer and progressive disease were treated by norethisterone. Estradiol and progesterone receptors were measured before starting treatment, and in three cases after 2 to 4.75 months of therapy. Partial remissions (greater than 50% decrease of all lesions) or objective improvements (decrease of 20-50%) were obtained in 7 patients, with a mean duration of 6.7 months. Stabilization of the lesions (no change or decrease of less than 20% in the size of the target lesion) was observed in 7 patients. No clear-cut correlation was found between clinical response and presence in biopsies of estradiol and progesterone receptors. However, absence of response occurred more frequently if tumors did not contain progesterone receptors.

Translation of uteroglobin mRNA in wheat-germ extract has yielded a precursor protein (pre-uteroglobin) containing an N-terminal extension of 21 amino acid residues. The sequence of this extension and that of the 50 N-terminal amino acid residues of uteroglobin have been determined.

When soluble steroid-receptor complexes are exposed to DNA-cellulose only activated complexes bind. The specificity of the binding was shown by its dependence on the presence of hormone during activation. However, prolonged incubation of non-activated steroid-receptor complexes with DNA-cellulose led to a progressive activation of these complexes. When the same hepatic cytosol containing heat-activated [3H]triamcinolone acetonide-receptor complexes was titrated by high concentrations of nuclei or DNA-cellulose the former bound 75% of the complexes, the later only 40%. This decreased binding was due on the one hand to a lower initial interaction between DNA-cellulose and activated complexes than between nuclei and these complexes and on the other hand to increased losses during washes when DNA-cellulose was used. For these reasons nuclei and not DNA-cellulose should be used when accurate measurements of the concentration of activated complexes are required. When only comparative data are needed DNA-cellulose may, however, be employed.

An antiserum specific for guinea pig progesterone-binding plasma protein (PBP) has been prepared. Using a very sensitive immunoenzymatic assay, PBP could be detected not only in pregnant guinea pig plasma (970 microgram/ml of plasma at day 40--60 of pregnancy), but also in the plasma of fetuses (2.77 micrograms/ml), umbilical arteries (1.79 micrograms/ml), umbilical vein (2.90 micrograms/ml), and in amniotic fluid (0.47 microgram/ml). The protein was also found in low concentration in the plasma of nonpregnant females (2.10 micrograms/ml) and males (1.56 micrograms/ml). The antiserum was used to search for immunological similarities between various steroid-binding proteins. No cross reaction was found with cavian or human corticosteroid-binding globulin and human sex steroid-binding plasma protein. There was no cross reaction between guinea pig PBP and PBP of other pregnant hystricomorphs (viscacha, degu, and coypu). Moreover, no evidence was found of an interaction between guinea pig uterine progesterone receptor and the anti-PBP antiserum.

Modifications of uterine blood flow are implicated in many important aspects of reproductive physiology and in several of their pathological disorders. These modifications are hormonally regulated but remain poorly understood, and various complex mechanisms have been proposed. The aim of this study was to investigate the presence and some characteristics of estrogen receptors (ER) and progesterone receptors (PR) in uterine blood vessels. Using monoclonal antibodies and immunocytochemistry we observed the presence of ER and PR in muscle cells (tunica media) of uterine arteries of rabbits and women. ER or PR immunoreactivity was not detected in the endothelium of uterine arteries nor in uterine capillaries or veins. Staining for both receptors was also present in arterial walls from the fallopian tube (isthmus and ampulla) and vagina but not in arteries of nonreproductive tissues (intestinal, renal, hepatic, femoral, and pulmonary arteries, aorta). PR immuno-staining was increased by estrogen in all cell types of the rabbit uterus, but the doses necessary to provoke an intense nuclear staining in uterine arteries were higher than those required for observing strong labeling in glandular, stromal, or myometrial cells. These results suggest that, contrary to many hypotheses previously put forward, sex steroid hormones may regulate uterine blood flow through a direct effect on uterine arterial walls.

Previous in vivo studies have shown that the rabbit progesterone receptor undergoes two phosphorylation reactions: one basal and a second one which is hormone-dependent. We report here on the presence and characteristics of a kinase activity found in receptor preparations highly purified by immunoaffinity chromatography. 1. This kinase activity is not due to the receptor molecule itself since the two proteins may be separated by several chromatographic and immunological methods. 2. The presence of the kinase in receptor preparations is not an artefact of the purification procedure. The kinase binds to the receptor as shown by coelution in immunoaffinity experiments and during various chromatographies. This interaction probably takes place in vivo and is not artefactually formed during solubilization of the receptor since the kinase also copurifies with receptors isolated from the uterine nuclei of progestin-treated rabbits. 3. This enzyme may be classified as a casein kinase since it readily phosphorylates the latter substrate (Km approximately equal to 0.15 mg/ml) and is not regulated by cyclic nucleotides, Ca2+ and calmodulin or phospholipids. Its classification as a casein kinase I or II is difficult since on the one hand it is inhibited by heparin, activated by polyamines and may use both ATP and GTP, but on the other hand it modifies only serine residues, and is not inhibited by heparin when the receptor itself is employed as a substrate. 4. The kinase which copurifies with the receptor does not mimic in vitro the effects of the hormone-dependent phosphorylation of the receptor observed in vivo: there is no enhancement of kinase activity by the hormone, and the phosphorylated receptor does not exhibit the characteristic "upshift" in its electrophoretic mobility. Thus either this kinase is not the enzyme responsible for the hormone-dependent receptor phosphorylation or, during purification, a factor has been lost which is necessary for retaining the hormone dependency of the reaction.

Two lambda gt11 clones containing fragments of cDNA encoding the rabbit progesterone receptor were isolated with the aid of monoclonal and monospecific polyclonal antireceptor antibodies. RNA gel blot analysis showed that the corresponding mRNA was approximately equal to 5900 nucleotides in size and present in the uterus, where its concentration was increased by estrogen treatment, and in the vagina. This mRNA was not detected in liver, in spleen, in intestine, and in kidney where the receptor protein is known to be absent or present in very small concentration. Cross-hybridizing clones were isolated from a lambda 10 library. The DNA was sequenced, and the primary structure of the progesterone receptor was deduced. It consists of 930 amino acids and contains a basic, cysteine-rich region (residues 568-645) with extensive homology to the glucocorticoid and estrogen receptors and the v-erbA oncogene protein. This region is followed by a C-terminal domain that is similar in size to the corresponding domains of the other steroid receptors and v-erbA and shows striking amino acid homology with the glucocorticoid receptor and significant homology with the estrogen receptor. In contrast, the region extending from the cysteine-rich segment toward the N terminus differed in size and amino acid sequence from that of the other receptors and v-erbA. This region had a high proline content in the progesterone receptor.

The messenger RNA (mRNA) coding for uteroglobin has been localized in the rabbit uterus and lung by in situ hybridization. Tissue sections fixed in ethanol-acetic acid were hybridized to the cloned complementary DNA probe labeled with tritium. The hybridization sites were detected by radioautography. Control experiments using [3H]pBR322 DNA demonstrated the specificity of the observed labeling. In the lung, uteroglobin mRNA, present in small concentrations, could be clearly visualized only after background was decreased by incubation of sections with S1 nuclease. In pregnant rabbit uterine horns, uteroglobin mRNA, visualized by silver grains, was found in the endometrial epithelium. The concentration was greater in the cells of glandular epithelium than in the cells of surface epithelium. Specific and intense labeling was spread through the cytoplasm. Practically all epithelial cells contained uteroglobin mRNA. Hybridization was very weak in the uterine epithelial cells of the nonpregnant rabbit. In the lung, a high degree of labeling occurred on the ciliated and bronchiolar cells of the epithelium of bronchi and bronchioles whereas the goblet cells remained unlabeled. Certain cells lining alveolar ducts and alveoli in the pulmonary parenchyma also showed a slight labeling. No differences in the labeling were observed in the lung of either pregnant or non-pregnant animals. There are several differences in the intensity and distribution of labeling between our hybridization experiments and previous studies involving immunocytochemical detection of uteroglobin protein. The latter technique thus probably not only reflects the pattern of synthesis of the protein but also depends on uteroglobin retention in the cells. Moreover, no evidence was found to bear out the hypothesis that some endometrial cells which contain uteroglobin do not synthesize this protein but take it up from endometrial fluid.